The field of this invention is human and/or veterinary vaccines and diagnostics, in particular vaccines comprising Pasteurella multocida neuraminidase and/or peptides having amino acid sequences derived therefrom and oligonucleotides useful as specific hybridization probes or specific polymerase chain reaction primers, wherein said vaccines are useful in protecting animals from infection and disease caused by P. multocida and wherein the probes or primers are useful in the diagnosis of infection by Pasteurella multocida and/or in the detection of pathogenic P. multocida. 
Pasteurella multocida is a gram-negative, oxidase positive rod-shaped bacterium which is a causative agent of human and animal diseases including fowl cholera, shipping fever in cattle, respiratory tract infection, abscesses and systemic infection in various animals. Humans can also be infected by P. multocida. This organism often colonizes mucosal tissue, especially in the respiratory system.
Five serotypes based on capsular antigen groupings have been described. Further typing is based on lipopolysaccharide (LPS) structure. Group A strains of P. multocida have a capsule which is mainly composed of hyaluronic acid. This capsule contributes to virulence by inhibiting phagocytosis and halogenation of bacterial proteins by the host defense system. The capsule is often lost during subcultures in vitro. Fimbriae are likely to mediate attachment to host tissue early in the infection process. Neuraminidase (sialidase) is an enzyme produced by most pathogenic strains of P. multocida; it is believed to contribute to infection and pathogenesis.
Various vaccines are available for P. multocida have been developed, with varying degrees of cross-protection for different serotypes and varying levels of effectiveness.
Because P. multocida infections pose a threat to the agricultural industry and because such infections result in significant economic losses, because veterinary care is expensive and because P. multocida can cause human infections as well, there is a longfelt need in the art for an effective, broad spectrum subunit vaccine to protect humans and animals against P. multocida. The present inventors believe that a vaccine comprising P. multocida neuraminidase and/or immunogenic peptides derived therefrom fulfill this need. In addition, there is a need for improved methods for diagnosis of P. multocida infections.
An object of the present invention is to provide immunogenic compositions comprising a neuraminidase derived from P. multocida or recombinantly expressed from a nucleotide sequence derived from P. multocida, which sequence encodes a neuraminidase (or NanH), having a predicted molecular mass of about 44 kDa as a mature protein. In a specifically exemplified P. multocida NanH protein, the protein is characterized by an amino acid sequence as given in SEQ ID NO:5, amino acids 1-412.
Whithin the scope of the present invention are methods for protecting animals, including without limitation, sheep, cattle, rabbits, cats, dogs, rodents such as mice, turkeys, chickens and other fowl, and humans, from infection and/or pathology caused at least in partby P. multocida, said method comprising the step of administering to said animal or human an immunogenic composition comprising the exemplified neuraminidase or other P. multocida neuraminidase with a primary structure similar (more than about 90% amino acid sequence identity) to the exemplified neuraminidase, and/or one or more peptides derived from one or more of the foregoing proteins or having amino acid sequence(s) taken from the amino acid sequence(s) of one or more of the foregoing proteins, wherein said peptide or protein, when used in an immunogenic composition in an animal, including a human, confers protection against infection by and/disease caused at least in part by P. multocida. As specifically exemplified, immunogenic peptides include VVMFDLRWKTASDQNRIDPG (SEQ ID NO: 1); MHGTWAAGTQNWYRDRLSY (SEQ ID NO:2); and HKHQVAIIRPGSGNAGAGYSSLAY (SEQ ID NO:3).
Substantially pure recombinant 47.4 to 50 kDa neuraminidase can be prepared after expression of a nucleotide sequence encoding neuraminidase in a heterologous host cell using the methods disclosed herein or from P. multocida outer membranes. Specifically exemplified partial neuraminidase amino acid sequences are given in Table 2.
As specifically exemplified herein, the nucleotide sequence encoding a mature P. multocida neuraminidase is given in SEQ ID NO:4, nucleotides 251 through 1486, exclusive of the signal peptide and stop codon. The complete coding sequence, including the N-terminal signal peptide of 21 amino acids is given in SEQ ID NO:4 from nucleotide 188 through 1486, exclusive of the stop codon. All synonymous coding sequences are within the scope of the present invention. The skilled artisan will understand that the coding or amino acid sequence of the exemplified neuraminidase protein can be used to identify and isolate additional, nonexemplified nucleotide sequences which will encode a functional protein of the same amino acid sequence as given in SEQ ID NO:5 from amino acid 1-412 or as given in SEQ ID NO:5 from xe2x88x9221 to 412 or an amino acid sequence of greater than 90% identity to either of the foregoing and having neuraminidase activity. Additional, partial neuraminidase coding sequences which identify other P. multocida sequences are given in Table 1 herein. The skilled artisan understands that it may be desirable to express the neuraminidase as a secreted protein; if so, it is known how to modify the exemplified coding sequence for the xe2x80x9cmaturexe2x80x9d neuraminidase, by adding a nucleotide sequence encoding a signal peptide appropriate to the host in which the sequence is expressed. The skilled artisan understands that it may be desirable to express the neuraminidase as a secreted protein; if so, it is known how to modify the exemplified coding sequence for the xe2x80x9cmaturexe2x80x9d neuraminidase, by adding a nucleotide sequence encoding a signal peptide appropriate to the host in which the sequence is expressed. When it is desired that the sequence encoding a neuraminidase protein be expressed, then the skilled artisan will operably link transcription and translational control regulatory sequences to the coding sequences, with the choice of the regulatory sequences being determined by the hose in which the coding sequence is to be expressed. With respect to a recombinant DNA molecule carrying a neuraminidase coding sequence, the skilled artisan will chose a vector (such as a plasmid or a viral vector) can be introduced into and which can replicate in the host cell. The host cell can be a bacterium, preferably Escherichia coli, or a yeast or a mammalian cell. Recombinant vectors carrying NanH coding sequences and recombinant host cells comprising same are also within the scope of the present invention.
The present invention also provides for fusion polypeptides comprising at least one epitope of a P. multocida neuraminidase, which is capable of providing full or partial protective immunity to an animal (or human) vaccinated with an effective amount of said fusion protein in an immunogenic composition. Homologous polypeptides may be fusions between two or more neuraminidase sequences. Likewise, heterologous fusions may be constructed which would exhibit a combination of properties or activities of the proteins from which they are derived. Fusion partners include, but are not limited to, a nontoxic fragment of cholera toxin, immunoglobulins, ubiquitin, bacterial xcex2-galactosidase, TrpE, protein A, xcex2-lactamase, alpha amylase, alcohol Dehydrogenase and yeast alpha mating factor [Godowski et al. (1988) Science 241:812-816]. Fusion proteins will typically be made by recombinant methods but may be chemically synthesized. Preferably, the NanH portion of such a fusion protein comprises the region encoded downstream of about nucleotide 1500 in SEQ ID NO:4.
Compositions and immunogenic preparations including but not limited to vaccines, comprising at least one P. multocida neuraminidase and/or a peptide derived therefrom, and a suitable carrier therefor are provide; preferably such immunogenic compositions further comprise an adjuvant. Such immunogenic compositions and vaccines are useful, for example, in immunizing an animal, including a human, against infection by and/or disease caused by P. multocida. The vaccine preparations comprise an immunogenic amount of a Pasteurella neuraminidase or an immunogenic peptide fragment or synthetic peptide of same. Such vaccines may comprise one or more neuraminidases in combination with another protein or other immunogen. By xe2x80x9cimmunogenic amountxe2x80x9d is meant an amount capable of eliciting the production of antibodies directed against one or more P. multocida neuraminidase (or one or more peptides whose amino acid sequence is derived from the foregoing protein) in an individual or animal to which the vaccine has been administered.
It is a further object of the present invention to provide primers and PCR-based methods for the diagnosis of a P. multocida infection or the detection of P. multocida cells in a sample, such as a biological sample including, but not limited to, respiratory system exudate, infected tissue, abscess-derived material, stool sample or tissue from a reservoir of infection such as the mouse. A sample may be taken from a suspected infected animal, for example in suspected outbreak of fowl cholera in chickens or turkeys, suspected shipping fever in sheep or cattle, a diseased rabbit, a cat or dog with an abscess or from a human potentially infected with P. multocida. Forward primer 5xe2x80x2-GCTTTGAATGGCAGTTTATATGTG-3xe2x80x2 (SEQ ID NO:6) and reverse primer 5xe2x80x2-TGAAGGAGCCGCTGTAGTCG-3xe2x80x2 (SEQ ID NO:7) (derived from the P. multocida R1913 nanH gene) are used to amplify a fragment of about 511 bp of a P. multocida nanH gene. The skilled artisan understands that alternative primers can also be designed using the nucleotide sequence information provided herein, taken with information readily accessible and well known to the art.
Additionally, the present invention provides for immunoassays for detection and/or diagnosis of Pasteurella multocida infection (or carrier state) in animals of humans. Such immunoassays contain antibodies specific for a neuraminidase of the present invention, where the neuraminidase is encoded by a nucleotide sequence having at least about 90% homology to SEQ ID NO:4, amino acids 1-412 and where the P. multocida neuraminidase in an infected or carrier animal has antigenic determinants in common with the specifically identified neuraminidase.